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COMPARATIVE STUDY ON THE VOLATILES’ COMPOSITION OF DICHOTOMA PALL. RHIZOME EXTRACTS WIRGINIA KUKULA-KOCH1*, KRYSTYNA SKALICKA-WOŹNIAK1, ELWIRA SIENIAWSKA1, JAROSŁAW WIDELSKI1, OTGONBATAAR URJIN2, PAWEŁ GŁOWNIAK1 1Department of Pharmacognosy with Medicinal Unit, Medical University of Lublin, 1 Chodzki, 20-093 Lublin, Poland 2School of Pharmacy, Health Sciences University of , 3 Zorig Street, 14210 Ulaanbaatar, Mongolia *corresponding author: [email protected]

Abstract The current study was performed to examine the qualitative and quantitative composition of the essential oil, n-hexane and ethanol extracts from the rhizomes of Iris dichotoma Pall. for their volatile content. The essential oil was particularly found to possess the most rich composition, while the latter extracts (n-hexane and ethanolic ones) consisted of various fatty acid esters. GC and GC-MS analyses of the oil resulted in the identification of 28 components. Among them myristic acid (13.67 %), methyl myristate (9.08 %), and α- terpineol (2.42 %) were found to be the dominant compounds. The qualitative composition of both n-hexane and ethanol extracts of Iris dichotoma Pall., obtained by pressurized liquid extraction, was similar and revealed the presence of 6 components, which were identified as natural products with methyl myristate as the major one (22.47 % and 10.18 % in ethanolic and n-hexane extracts, respectively).

Rezumat Studiul prezentat în acest articol a fost efectuat pentru a examina compozitia calitativă și cantitativă a uleiului volatil, a extractelor n-hexanic și etanolic obţinute din rizomi de Iris dichotoma Pall. în ceea ce privește conținutul lor volatil. S-a constatat că uleiul volatil are cea mai bogată compoziție, deși extractele finale (n-hexan și cele etanolice) constau din diverși esteri ai acizilor grași. Prin analizele GC și GC-MS ale uleiului s-au identificat 28 de compuși. Dintre acestea, acidul miristic (13,67%), metilmiristatul (9,08%) și α -terpineolul (2,42%) s-au dovedit a fi compusii dominanți. Compoziția calitativă a extractelor n-hexanic și etanolic, obținute prin metoda extracției cu lichide presurizate, a fost similară și a evidenţiat prezența a 8 componente. Şase dintre ele au fost identificate ca produși naturali, având miristatul de metil drept component major (22,47% și 10,18% în extractul etanolic și, respectiv, n-hexanic).

Keywords: Iris dichotoma, volatile oil, terpenes, GC-MS analysis.

Introduction Iris species belong to the plants widely distributed around the globe. They grow world-wide in tropical and temperate regions of the world, with

FARMACIA, 2014, Vol. 62, 5 875 the highest diversity occurring in South Africa, followed by South America, Europe and temperate regions of [6]. Approximately 300 species of these colourfully flowering plants have been described so far [7]. Iris species have an immense medicinal importance and are used in the treatment of cancer, inflammation, bacterial, and viral infections [10, 18, 19]. Numerous scientific papers treating on the variety of their pharmacological activities and evoking the presence of isoflavones, flavones, terpenoids, xanthones, or simple phenolics in the extracts have been published within recent years [7, 11, 13, 18]. Iris dichotoma Pall. is a perennial herb that is native to the north . It is an elegant herb, 40–60 cm long with underground rhizomes. The leaves are 1.5–3.5 cm wide, 15–35 cm long, sword-shaped, slightly curved, with no midvein present. The has one green flower stalk dichotomously branched rising from a rhizome. The flowers are 12–15 cm in diameter, violet, pale blue, or cream with purplish brown markings [24]. Rhizomes of I. dichotoma, called Bai-Sheganare commonly used in Chinese folk medicine for clearing heat and detoxification, phlegm elimination, swelling, and sore throat [25]. The main active components in I. dichotoma are phenolic compounds, namely flavonoid or isoflavonoid aglycones and their corresponding glycosides. Previous phytochemical studies indicated that the flavonoids of I. dichotoma include wogonin, rhamnazin, isovitexin, hispidulin, rhamnocitrin and tamarixetin-7-glucoside, whereas the isoflavonoids of I. dichotoma include irisflorentin, dichotomitin, irigenin, genistin, tectorigenin, tectoridin, iristectorigenin A, iristectorin A and iristectorin B [11, 14, 16]. Although the polyphenolic compounds of I. dichotoma are well described, the chemical composition of its essential oil is lacking. In the recent scientific literature only few studies treat on the composition of essential oil obtained from other Iris species. Iris nigricans Dinsm. – endemic to Jordan – was found to possess significant amounts of aliphatic hydrocarbons in the essential oil, but also monoterpene hydrocarbons represented by α- and β-pinenes [4]. Syrian species of iris rhizomes were found to produce myristic acid as the main component of the terpene fractions. Furthermore, lauric acid, decanoic acid, palmitic acid and its methyl ester, octadecanoic acid methyl ester, and elaidic acid metyl ester were identified as sub major compounds. Stearic acid, methyl oleate, palmitic acid and 1,8-cineole belong to the major constituents of Iris pseudoacorus essential oil [5]. The above mentioned results confirm the diversity of essential oils’ composition depending on the investigated species.

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In this paper, the essential oil as well as n-hexane and ethanolic extracts of I. dichotoma rhizomes were analysed.

Materials and Methods Plant material Rhizomes of Iris dichotoma Pall. () were collected in Mongolia Dornot province, Bayandun Ulaanbataar and identified by Otgonbataar Urjin. A voucher specimen has been deposited in the Chair and Department of Pharmacognosy with Medicinal Plant Unit (WID10001). Preparation of the extracts • Hydrodistillation in a Deryng apparatus 100 g of dried rhizomes of Iris dichotoma Pall. were coarsely powdered, transferred to a round-bottomed flask and submitted to steam distillation process with 250 mL of water in a Deryng-type apparatus for 4 hours. The obtained essential oil was dried over anhydrous sodium sulphate and stored in amber glass at 4°C. • Extraction in a Pressurized Liquid Extractor (PLE) Pressurized Liquid Extraction was performed on a Dionex 100 apparatus (Dionex, Sunnyvale, USA). 5 g of rhizomes were placed in a 34 mL stainless steel extraction cell and extracted with n-hexane. The detailed extraction conditions were set as follows: extraction temperature: 60°C, static time: 15 min, flush volume: 100%, purge time: 80 sec, number of cycles: 3. The operating pressure was ranging around 105 bar. The same procedure was repeated when ethanol was used as a solvent. The obtained extracts were subsequently evaporated to dryness under vacuum on a water bath at 25°C, re-dissolved in 3 mL of n-pentane, filtered through a nylon membrane filter (nominal pore size: 45 µm) and transferred to an amber glass vial. Reagents All solvents used were of analytical grade and were purchased from the Polish Reagents (POCH, Gliwice, Poland). Helium 5.0 used for GC-MS analysis was 99.999% pure (PGNiG, Poland). GC-MS GC-MS was performed with a Shimadzu GC-2010 Plus gas chromatography instrument coupled to a Shimadzu QP2010 Ultra mass spectrometer. Compounds were separated on a fused silica capillary column ZB-5 MS (30 m, 0.25 mmi.d.) with a film thickness of 0.25 µm (Phenomenex). The following oven temperature program was initiated at 50oC [17], held for 3 min, then increased at the rate of 5oC min-1 to 250oC, held for 15 min. Injector, interface and ion source were kept at 250, 250 and

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220oC, respectively. Split injection (1 µL) was conducted with a split ratio of 1:20 and helium was used as carrier gas of 1.0 mL*min-1 flow-rate. The spectrometers were operated in electron-impact (EI) mode, the scan range was 40–500 amu, the ionization energy was 70 eV and the scan rate was 0.20 s per scan. The retention indices were determined in relation to a homologous series of n-alkanes (C8-C24) under the same operating conditions. Compounds were identified using a computer-supported spectral library [1], mass spectra of reference compounds, as well as MS data from the literature [2, 3].

Results and Discussion A significant number of Iris species have been reported for their medicinal properties so far. The plants were used in Asian traditional medicine to treat cough, sore throat or asthma [12, 21, 23]. Currently they have been reported to possess notable antimicrobial, antifungal, chemopreventive, anti-tubercular and antioxidant properties [8, 22] mainly due to the isoflavone content, although Deng et al. [8] confirmed marked antibacterial and antifungal properties of the essential oil derived from Iris bulleyana. The terpene composition of Iris dichotoma has not been reported so far. As the plant is widely used in traditional medicine of Mongolia and China until nowadays, the authors found it reasonable to identify the dominant volatiles, which presence may influence its overall pharmacological activity. The major constituents of the essential oil obtained from Iris dichotoma rhizomes are shown in the Table I. while the composition of n- hexane and ethanol extracts is presented in the Table II. 28 compounds representing 39.52% of essential oil were identified. Myristic acid (13.67%) and methyl myristate (9.08%) were the most abundant components. The others were α-terpineol (2.42%), hexadecanoic acid methyl ester (1.86%), brachyloxide (1.62%), n-decanoic acid (1.22%) and geranyl acetone (1.04%). The following chemical components occurred in the analyzed essential oil in smaller amounts: eudesm-4(15)en-7-ol (0.92%), isoneral (0.82%), 1-epi-cubenol (0.77%), tetradecanal (0.77%), octanoic acid (0.58%), nonanoic acid (0.52%), trans anethol (0.48%), δ-cadinene (0.44%), trans-geraniol (0.43%), α-caracorene (0.41%), cymen-9-ol (0.36%), linalool (0.33%), α-muurolene (0.31 %), α-cadinol (0.27%), cis-verbenol and 2,4- decadienal (0.2%), capric acid methyl ester and geranial (0.16%) and m- eugenol (0.11%) (Table I).

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Table I Chemical composition of the volatile compounds of Iris dichotoma rhizomes obtained by hydrodistillation of essential oil (RRI – Relative Retention Index). No Retention RRI RRI Area Compound time [min] NIST MF % 1 Linalool 13.24 1105 1086 0.33 2 cis-Verbenol 14.54 1148 1132 0.20 3 Isoneral 14.65 1152 1140 0.82 4 Octanoic acid 15.80 1189 1156 0.58 5 Cymen-9-ol 15.99 1196 1157 0.36 6 α-Terpineol 16.19 1203 1176 2.42 7 trans-Geraniol 17.74 1256 1235 0.43 8 α-Citral 18.19 1272 1244 0.16 9 Nonanoic acid 18.53 1284 1263 0.52 10 trans-Anethol 18.72 1293 1262 0.48 11 2,4-Decadienal 19.63 1324 1270 0.2 12 Capric acid methyl ester (methyl decanoate) 19.72 1327 1300 0.16 13 m-Eugenol 20.59 1359 1346 0.11 14 n-Decanoic acid 21.29 1385 1347 1.22 15 Tetradecanal 22.05 1414 0.77 16 Geranyl acetone 23.02 1452 1430 1.04 17 2-Tridecanone 24.22 1500 1477 0.3 18 α-Muurolene 24.37 1505 1496 0.31 19 δ-Cadinene 24.86 1526 1520 0.44 20 α-Caracorene 25.44 1550 1527 0.41 21 Brachyloxide 26.80 1607 1599 1.62 22 1-Epi-cubenol 27.52 1638 1623 0.77 23 Eudesm-4(15)en-7-ol 27.88 1654 1643 0.92 24 α-Cadinol 28.20 1668 1643 0.27 25 Methyl myristate (myristic acid methyl ester) 29.58 1730 1700 9.08 26 Farnesal 29.88 1744 1707 0.07 27 Myristic acid (tetradecanoic acid) 30.83 1788 13.67 28 Hexadecanoic acid methyl ester 33.80 1932 1.86 MF = Mass Finder, NIST = National Institute of Standards and Technology

6 compounds representing 15.82 and 35.36 percent of hexane and ethanol extracts from Iris dichotoma rhizomes, respectively, were identified. Methyl myristate was the most abundant component in both hexane and ethanol extracts (10.18% and 22.47%). In the case of hexane extract methyl palmitate (3.15%) and methyllinoleate (1.21%) were identified. Presence of methoxyeugenol (0.46%), methyl oleate and methyl stearate (0.41%) was confirmed in hexane extract in small amounts. The other components of ethanol extract were methyl palmitate (6.99%), methyl linoleate (2.76%),

FARMACIA, 2014, Vol. 62, 5 879 methoxyeugenol (1.23%) and methyl oleate (1.0%). Methyl stearate (0.91%) occurred in ethanol extract in small amounts (Table II). Table II Chemical composition of the volatile compounds of Iris dichotoma rhizomes in hexane and ethanol extracts (RRI – Relative Retention Index) No Retention RRI RRI Area % Area % Compound time [min] NIST MF (Hexane) (Ethanol) 1 4-Allyl-2,6-dimethoxyphenol (methoxyeugenol) 26.72 1604 1561 0.46 1.23 2 Methyl myristate 29.59 1730 1700 10.18 22.47 3 Methyl palmitate 33.80 1932 1901 3.15 6.99 4 Methyl linoleate 37.02 2099 2046 1.21 2.76 5 Methyl oleate 37.14 2106 2082 0.41 1.00 6 Methyl stearate 37.65 2134 2104 0.41 0.91 MF = Mass Finder, NIST = National Institute of Standards and Technology

The qualitative composition of both n-hexane and ethanol extracts of Iris dichotoma varied from the hydrodistilled essential oil. The former extracts contained significant amounts of long-chained constituents, which prevailed over simple terpenes - so abundant in the essential oil. Both pressurised liquid extraction (PLE) extracts contained methoxyeugenol which was absent in the essential oil. Furthermore, a variety of long-chained esters of fatty acids appeared in the GC spectrum after pressurized liquid extraction. Composition of Iris dichotoma essential oil differed from the one described in the case of other species [4, 5, 20]. 1,8-cineole was not present in the investigated rhizomes. Moreover, pinenes - major components of Jordan’s Iris spp. - were absent in the studied samples. Myristic acid and its methyl ester were identified in the essential oil of the Mongolian species and constituted the major products of the hydrodistilled oil, similarly to the formerly discussed Syrian species of the same genus [5]. Based on these observations, it can be concluded, that Iris dichotoma is characterized by a distinguishing composition of terpenes. Presented results confirmed marked intra-species differences. Both n-hexane and ethanol extracts contained fatty acids’ esters which are common among Iris rhizomes. Esters of palmitic, stearic, oleic or linoleic acids were found in Iris dichotoma, but were also described in other Iris species.

Conclusions Rhizomes of Iris dichotoma Pall. are characterized by a high content of essential oil, distinguished by a rich chemical composition. Among 28

880 FARMACIA, 2014, Vol. 62, 5 volatile components of the essential oil - myristic acid, methyl myristate and α-terpineol were dominant. Chemical composition of volatiles varied depending on the extraction conditions applied. The content of ethanol and n-hexane extracts obtained by means of PLE extraction revealed the presence of long-chained fatty acids’ esters with methyl myristate and methyl palmitate as the major ones.

Acknowledgements The authors would like to thank Shim-Pol A.M. Borzymowski Company, sole representative of Shimadzu and Phenomenex in Poland, for GC-MS equipment and ZB-5 MS column. The authors have declared no conflict of interest.

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